2.1. Materials

[³H]-labeled estrone sulfate was acquired from PerkinElmer (Waltham, MA). COS-7 cells were acquired from American Type Culture Collection (Manassas, VA). Membrane-impermeable biotinylation reagent NHS-SS-biotin, streptavidin-agarose beads and protein G-agarose beads were acquired from Pierce (Rockford, IL). cDNA for human Nedd4–2 was kindly provided by Dr. Peter M. Snyder of the College of Medicine, University of Iowa (Iowa City, IA). cDNAs for mouse sgk2 (wild-type sgk2, inactive sgk2 (IN-sgk2) and constitutive active sgk2 (CA-sgk2) were kindly provided by Dr. Alan C. Pao from Department of Medicine, Stanford University (Stanford, CA). Mouse anti-Myc antibody (9E10) was acquired from Roche (Indianapolis, IN). Rabbit anti-Nedd4–2, was purchased from Abcam (Cambridge, MA). Rabbit anti-P-Nedd4–2 (Serine 327) antibody was acquired from Cell Signaling (Danvers, MA). Mouse anti‐β‐actin was acquired from Santa Cruz (Santa Cruz, CA). Nedd4–2-specific siRNA oligonucleotides (Silencer® Select, identification number s23570) and negative control siRNA oligonucleotides (Silencer® Select, catalog number 4390843) were acquired from Ambion (Grand Island, NY). Insulin and all other reagents were acquired from Sigma-Aldrich (St. Louis, MO).